Duplex DNA Destabilization by Type V CRISPR-Cas Nucleases during Interrogation of DNA

Abstract

The prokaryotic CRISPR-Cas effector complexes locate their target sites by scanning and interrogating the genomic DNA. The type II and type V CRISPR-Cas nucleases cleave double-stranded DNA bearing sequences complementary to guide segments of CRISPR RNAs (crRNA) flanked by conserved PAM sequences; they have been successfully used for genome editing in various organisms. Unpairing of a short PAM-proximal segment of DNA protospacer is a critical event that may determine the overall rate of target location. We sought to elucidate mechanistic details of initial DNA interrogation steps by type V CRISPR-Cas nucleases Cas12a and Cas12b (also known as Cpf1 and C2c1, respectively). Using fluorometric and biochemical assays, we studied interactions of Cas12a and Cas12b effectors with a set of model DNA substrates that mimic likely early intermediates on the pathway to the final R-loop complexes. Consistent with reported data on the mechanism of DNA interrogation by type II Cas9 nuclease, we find that binding of type V effectors to PAM favors separation of several adjacent to PAM DNA protospacer base pairs even in the absence of pairing between the crRNA guide and the protospacer. The separation is mediated in part by effector affinity for non-target DNA strand nucleotides in a single-stranded conformation. In contrast, effector interactions with the nearest to PAM target DNA strand nucleotides are energetically unfavorable in the type V early intermediate complexes, presumably due to a steric clash between the protein and DNA backbones. We suggest that these unfavorable interactions destabilize the DNA duplex and thus facilitate the DNA interrogation process. We discuss the similarities and differences between the mechanisms of DNA interrogation by Cas9, Cas12a and Cas12b effectors.